Trisomy 21 mid-trimester amniotic fluid induced pluripotent stem cells maintain genetic signatures during reprogramming: implications for disease modeling and cryobanking.

Trisomy 21 is the most common chromosomal abnormality and is associated primarily with cardiovascular, hematological, and neurological complications. A robust patient-derived cellular model is necessary to investigate the pathophysiology of the syndrome because current animal models are limited and access to tissues from affected individuals is ethically challenging. We aimed to derive induced pluripotent stem cells (iPSCs) from trisomy 21 human mid-trimester amniotic fluid stem cells (AFSCs) and describe their hematopoietic and neurological characteristics. Human AFSCs collected from women undergoing prenatal diagnosis were selected for c-KIT(+) and transduced with a Cre-lox-inducible polycistronic lentiviral vector encoding SOX2, OCT4, KLF-4, and c-MYC (50,000 cells at a multiplicity of infection (MOI) 1-5 for 72 h). The embryonic stem cell (ESC)-like properties of the AFSC-derived iPSCs were established in vitro by embryoid body formation and in vivo by teratoma formation in RAG2(-/-), γ-chain(-/-), C2(-/-) immunodeficient mice. Reprogrammed cells retained their cytogenetic signatures and differentiated into specialized hematopoietic and neural precursors detected by morphological assessment, immunostaining, and RT-PCR. Additionally, the iPSCs expressed all pluripotency markers upon multiple rounds of freeze-thawing. These findings are important in establishing a patient-specific cellular platform of trisomy 21 to study the pathophysiology of the aneuploidy and for future drug discovery.

Gene correction of induced pluripotent stem cells derived from a murine model of X-linked chronic granulomatous disorder.

Gene therapy presents an attractive alternative to allogeneic haematopoietic stem cell transplantation (HSCT) for treating patients suffering from primary immunodeficiency disorder (PID). The conceptual advantage of gene correcting a patient's autologous HSCs lies in minimizing or completely avoiding immunological complications arising from allogeneic transplantation while conferring the same benefits of immune reconstitution upon long-term engraftment. Clinical trials targeting X-linked chronic granulomatous disorder (X-CGD) have shown promising results in this context. However, long-term clinical benefits in these patients have been limited by issues of poor engraftment of gene-transduced cells coupled with transgene silencing and vector induced clonal proliferation. Novel vectors incorporating safety features such as self-inactivating (SIN) mutations in the long terminal repeats (LTRs) along with synthetic promoters driving lineage-restricted sustainable expression of the gp91phox transgene are expected to resolve the current pitfalls and require rigorous preclinical testing. In this chapter, we have outlined a protocol in which X-CGD mouse model derived induced pluripotent stem cells (iPSCs) have been utilized to develop a platform for investigating the efficacy and safety profiles of novel vectors prior to clinical evaluation.

Gene therapy for PIDs: progress, pitfalls and prospects.

Substantial progress has been made in the past decade in treating several primary immunodeficiency disorders (PIDs) with gene therapy. Current approaches are based on ex-vivo transfer of therapeutic transgene via viral vectors to patient-derived autologous hematopoietic stem cells (HSCs) followed by transplantation back to the patient with or without conditioning. The overall outcome from all the clinical trials targeting different PIDs has been extremely encouraging but not without caveats. Malignant outcomes from insertional mutagenesis have featured prominently in the adverse events associated with these trials and have warranted intense pre-clinical investigation into defining the tendencies of different viral vectors for genomic integration. Coupled with issues pertaining to transgene expression, the therapeutic landscape has undergone a paradigm shift in determining safety, stability and efficacy of gene therapy approaches. In this review, we aim to summarize the progress made in the gene therapy trials targeting ADA-SCID, SCID-X1, CGD and WAS, review the pitfalls, and outline the recent advancements which are expected to further enhance favourable risk benefit ratios for gene therapeutic approaches in the future.

Human mid-trimester amniotic fluid stem cells cultured under embryonic stem cell conditions with valproic acid acquire pluripotent characteristics.

Human mid-trimester amniotic fluid stem cells (AFSC) have promising applications in regenerative medicine, being broadly multipotent with an intermediate phenotype between embryonic (ES) and mesenchymal stem cells (MSC). Despite this propluripotent phenotype, AFSC are usually cultured in adherence in a serum-based expansion medium, and how expansion in conditions sustaining pluripotency might affect their phenotype remains unknown. We recently showed that early AFSC from first trimester amniotic fluid, which endogenously express Sox2 and Klf4, can be reprogrammed to pluripotency without viral vectors using the histone deacetylase inhibitor valproic acid (VPA). Here, we show that mid-trimester AFSC cultured under MSC conditions contained a subset of cells endogenously expressing telomerase, CD24, OCT4, C-MYC, and SSEA4, but low/null levels of SOX2, NANOG, KLF4, SSEA3, TRA-1-60, and TRA-1-81, with cells unable to form embryoid bodies (EBs) or teratomas. In contrast, AFSC cultured under human ESC conditions were smaller in size, grew faster, formed colonies, upregulated OCT4 and C-MYC, and expressed KLF4 and SOX2, but not NANOG, SSEA3, TRA-1-60, and TRA-1-81. Supplementation with VPA for 5 days further upregulated OCT4, KLF4, and SOX2, and induced expression of NANOG, SSEA3, TRA-1-60, and TRA-1-81, with cells now able to form EBs and teratomas. We conclude that human mid-trimester AFSC, which may be isolated autologously during pregnancy without ethics restriction, can acquire pluripotent characteristics without the use of ectopic factors. Our data suggest that this medium-dependant approach to pluripotent mid-trimester AFSC reflects true reprogramming and not the selection of prepluripotent cells.

Brief report: self-organizing neuroepithelium from human pluripotent stem cells facilitates derivation of photoreceptors.

Retinitis pigmentosa, other inherited retinal diseases, and age-related macular degeneration lead to untreatable blindness because of the loss of photoreceptors. We have recently shown that transplantation of mouse photoreceptors can result in improved vision. It is therefore timely to develop protocols for efficient derivation of photoreceptors from human pluripotent stem (hPS) cells. Current methods for photoreceptor derivation from hPS cells require long periods of culture and are rather inefficient. Here, we report that formation of a transient self-organized neuroepithelium from human embryonic stem cells cultured together with extracellular matrix is sufficient to induce a rapid conversion into retinal progenitors in 5 days. These retinal progenitors have the ability to differentiate very efficiently into Crx(+) photoreceptor precursors after only 10 days and subsequently acquire rod photoreceptor identity within 4 weeks. Directed differentiation into photoreceptors using this protocol is also possible with human-induced pluripotent stem (hiPS) cells, facilitating the use of patient-specific hiPS cell lines for regenerative medicine and disease modeling.

Valproic acid confers functional pluripotency to human amniotic fluid stem cells in a transgene-free approach.

Induced pluripotent stem cells (iPSCs) with potential for therapeutic applications can be derived from somatic cells via ectopic expression of a set of limited and defined transcription factors. However, due to risks of random integration of the reprogramming transgenes into the host genome, the low efficiency of the process, and the potential risk of virally induced tumorigenicity, alternative methods have been developed to generate pluripotent cells using nonintegrating systems, albeit with limited success. Here, we show that c-KIT+ human first-trimester amniotic fluid stem cells (AFSCs) can be fully reprogrammed to pluripotency without ectopic factors, by culture on Matrigel in human embryonic stem cell (hESC) medium supplemented with the histone deacetylase inhibitor (HDACi) valproic acid (VPA). The cells share 82% transcriptome identity with hESCs and are capable of forming embryoid bodies (EBs) in vitro and teratomas in vivo. After long-term expansion, they maintain genetic stability, protein level expression of key pluripotency factors, high cell-division kinetics, telomerase activity, repression of X-inactivation, and capacity to differentiate into lineages of the three germ layers, such as definitive endoderm, hepatocytes, bone, fat, cartilage, neurons, and oligodendrocytes. We conclude that AFSC can be utilized for cell banking of patient-specific pluripotent cells for potential applications in allogeneic cellular replacement therapies, pharmaceutical screening, and disease modeling.

Functional human artificial chromosomes are generated and stably maintained in human embryonic stem cells.

We present a novel and efficient non-integrating gene expression system in human embryonic stem cells (hESc) utilizing human artificial chromosomes (HAC), which behave as autonomous endogenous host chromosomes and segregate correctly during cell division. HAC are important vectors for investigating the organization and structure of the kinetochore, and gene complementation. HAC have so far been obtained in immortalized or tumour-derived cell lines, but never in stem cells, thus limiting their potential therapeutic application. In this work, we modified the herpes simplex virus type 1 amplicon system for efficient transfer of HAC DNA into two hESc. The deriving stable clones generated green fluorescent protein gene-expressing HAC at high frequency, which were stably maintained without selection for 3 months. Importantly, no integration of the HAC DNA was observed in the hESc lines, compared with the fibrosarcoma-derived control cells, where the exogenous DNA frequently integrated in the host genome. The hESc retained pluripotency, differentiation and teratoma formation capabilities. This is the first report of successfully generating gene expressing de novo HAC in hESc, and is a significant step towards the genetic manipulation of stem cells and potential therapeutic applications.

Generation of functional neutrophils from a mouse model of X-linked chronic granulomatous disorder using induced pluripotent stem cells.

Murine models of human genetic disorders provide a valuable tool for investigating the scope for application of induced pluripotent stem cells (iPSC). Here we present a proof-of-concept study to demonstrate generation of iPSC from a mouse model of X-linked chronic granulomatous disease (X-CGD), and their successful differentiation into haematopoietic progenitors of the myeloid lineage. We further demonstrate that additive gene transfer using lentiviral vectors encoding gp91(phox) is capable of restoring NADPH-oxidase activity in mature neutrophils derived from X-CGD iPSC. In the longer term, correction of iPSC from human patients with CGD has therapeutic potential not only through generation of transplantable haematopoietic stem cells, but also through production of large numbers of autologous functional neutrophils.

Nonintegrating lentivector vaccines stimulate prolonged T-cell and antibody responses and are effective in tumor therapy.

Lentiviral vectors (lentivectors) are effective for stimulation of cell-mediated and humoral immunity following subcutaneous and intramuscular immunization. However, lentivector genome integration carries a risk of perturbation of host gene expression. Here, we demonstrate that lentivectors with multiple mutations that prevent integration are also effective immunogens. First, systemic CD8(+) T-cell responses to the model antigen ovalbumin were detected following subcutaneous injection of nonintegrating lentivectors. Transfer of transgenic OT1 T cells demonstrated that antigen presentation persisted for at least 30 days. Furthermore, an enhanced CD8(+) T-cell response, peaking at 7 days, was stimulated by coexpression of p38 MAP kinase or an NF-kappaB activator from the same vector. Second, we demonstrated systemic CD8(+) T-cell and antibody responses to the secreted hepatitis B virus (HBV) surface antigen expressed from a nonintegrating lentivector injected intramuscularly. The induction, specificity, and kinetics of antibody production closely mimicked those of natural HBV infection. In this case, both the vector genome and the immune response were maintained for at least 2 months. Together, our data indicate that nonintegrating lentivectors can be employed to generate effective vaccines.

A HIV-2-based self-inactivating vector for enhanced gene transduction.

The employment of HIV-1-based vectors in clinical trials is controversial mainly due to the lethal nature of the virus. HIV-2 is less pathogenic in nature and therefore is likely to be safer for vector design and production. We developed HIV-2-based self-inactivating vectors in which 520 bp out of 554 bp of the viral U3 was deleted. Interestingly, high titers were obtained only when an exogenous promoter was used to drive expression of viral RNA. It was found that the vectors could target a wide range of mammalian cell types including primary neuronal cells and could yield long term expression. It is also noteworthy that the HIV-2 vectors could be effectively cross-packaged into HIV-1 core, which might provide for enhanced safety by reducing the recombination potential of the system.

Proviral progeny of heterodimeric virions reveal a high crossover rate for human immunodeficiency virus type 2.

Human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS in humans, exhibits a very high rate of recombination. Bearing in mind the significant epidemiological and clinical contrast between HIV-2 and HIV-1 as well as the critical role that recombination plays in viral evolution, we examined the nature of HIV-2 recombination. Towards this end, a strategy was devised to measure the rate of crossover of HIV-2 by evaluating recombinant progeny produced exclusively by heterodimeric virions. The results showed that HIV-2 exhibits a crossover rate similar to that of HIV-1 and murine leukemia virus, indicating that the extremely high rate of crossover is a common retroviral feature.

High rate of genetic recombination in murine leukemia virus: implications for influencing proviral ploidy.

A significant difference in the recombination rates between human immunodeficiency virus type 1 (HIV-1) and the gammaretroviruses was previously reported, with the former being 10 to 100 times more recombinogenic. It is possible that preferential copackaging of homodimers in the case of gammaretroviruses, like murine leukemia virus (MLV), led to the underestimation of their rates of recombination. To reexamine the recombination rates for MLV, experiments were performed to control for nonrandom copackaging of viral RNA, and it was found that MLV and HIV-1 exhibit similar crossover rates. The implications for control of proviral ploidy and preferential recombination during minus-strand DNA synthesis are discussed.